CHAPTER 23 BIOTECHNOLOGY - ilmkidunya.com

CHAPTER

23 BIOTECHNOLOGY

Animation 23 : Biotechnology

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23.Biotechnology eLearn.Punjab

Since Mendel’s work was rediscovered in 1900, geneticists have made startling advances

which have led to a new era of DNA technology. Modem techniques enable

desired substance, for example insulin. Not very long ago, people with insulin dependent

they receive human insulin, a product of biotechnology. Since the 1980s, biotechnology

has produced drugs and vaccines to curb human illnesses.

Genetically, engineered bacteria have been used to clean up environmental pollutants,

increase the fertility of the soil, and kill insect pests. Biotechnology also extends beyond

multicellular organisms. It is now possible to alter the genotype and subsequently the

phenotype of plants and animals. Indeed, gene therapy in humans, attempting to repair

a faulty gene is already undergoing clinical trials. There are those who are opposed to

manipulation of genes for any reason. Although, there have been no ill efects as yet, they fear the possibility of health and ecological repercussions in the future.

Cloning of a gene

Produces many identical copies. Recombinant DNA technology is used when a very

large quantity of a gene is required. The use of polymerase chain reaction (PCR) creates

a lesser number of copies within a laboratory test tube.

Recombinant DNA Technology

Recombinant DNA technology popularly known as genetic engineering aims at

synthesizing recombinant DNA which contains DNA from two diferent sources. In order to produce recombinant DNA, the following are required:

1. Gene of interest, which is to be cloned.

2. Molecular scissors to cut out the gene of interest.

3. Molecular carrier or vector, on which gene of interest could be placed.

4. The gene of interest alongwith the vector is then introduced into an expression

system, as a result of which a speciic product is made.

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How to get a gene?

There are three possible ways to get the gene of interest.

(a) to isolate it from the chromosome

(b) to synthesize it chemically, and

(c) to make it from mRNA

Genes can be isolated from the chromosomes by cutting the chromosomes on the

lanking sites of the gene using special enzymes known as restriction endonucleases. If, however, the genes are small, they can also be synthesized in the laboratory.

Another very common method of getting the gene is to synthesize it in the laboratory

from messenger RNA, using reverse transcriptase. This DNA molecule is called

complementary DNA (cDNA).

Molecular Scissors: Restriction Endonucleases

These are natural enzymes of bacteria, which they use for their own protection against

viruses. The restriction enzyme cuts down the viral DNA, but does no harm to the

bacterial chromosofhe. They are called restriction enzymes because they restrict the

growth of viruses. In 1970, Hamilton O. Smith, at Johns Hopkins University, isolated the

irst restriction enzyme. Bacteria produce a variety of such restriction enzymes, which cut the DNA at very speciic sites characterized by speciic sequence of four or six nucleotides arranged symmetrically in the reverse order. Such sequences are known

as palindromic sequences. So far more than 400 such enzymes have been isolated out

of which about 20 are frequently used in recombinant DNA technology.

EcoRl, a commonly used restriction enzyme, cuts double-stranded DNA when it has

this sequence of bases at the cleavage site (Fig. 23.1). Notice there is now a gap into

which a piece of foreign DNA can be placed, if it ends in bases complementary to those

exposed by the restriction enzyme. The single stranded but complementary ends of the

two DNA molecules are called “sticky ends” because they can bind by complementary

base pairing. They, therefore, facilitate the insertion of foreign DNA into vector DNA.

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Molecular Carrier: Vector

To make recombinant DNA, one often begins by selecting a vector, the means by

which recombinant DNA is introduced into a host cell. One common type of vector

is a plasmid. Plasmids were discovered by investigators studying the sex life of the

intestinal bacterium Escherichia coli.

Plasmids are natural extra-chromosomal circular DNA molecules which carry genes for

antibiotic resistance and fertility etc. One of the plasmids discovered earlier pSC 101 has

antibiotic resistance gene for tetracycline, whereas pBR 322 has antibiotic resistance

genes for tetracycline as well as ampicillin. Inserting gene of interest in tetracycline

resistant gene of plasmid pBR 322 would enable separating out colonies of bacteria in

a medium containing ampicillin and vice versa.

Fig, 23.1 Restriction enzyme ECoRl, cuts this speciic sequence of nucleotides in such a way that sticky ends are produced.

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Recombinant DNA

For preparation of a recombinant DNA, the plasmid is cut with the same enzyme which

was used for isolation of the gene of interest (Fig. 23.2). The gene of interest (insulin)

is then joined with the sticky ends produced after cutting the plasmid with the help of

another special enzyme knqwn as DNA ligase. This enzyme seals the foreign piece of

DNA into the vector. Now the two diferent pieces of DNA have been joined together, which is now known as recombinant DNA or chimaeric DNA

Animation 23.1: Recombinant DNA

Source & Credit:Pinterest

https://www.pinterest.com/pin/103512491408305996/

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Fig. 23.2 Cloning of a gene.

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Expression of the Recombinant DNA

A clone can be a large number of molecules (i.e. cloned genes) or cells (i.e. cloned

bacteria) or organisms that are identical to an original specimen. Fig. 23.3 compares the

use of a plasmid and a virus to clone a gene. Bacterial cells take up recombinant plasmid,

especially, if they are treated with calcium chloride to make them more permeable.

Thereafter, as the cell reproduces, a bacterial clone forms and each new cell contains at

least one plasmid. Therefore, each of the bacteria contains the gene of interest, which

will express itself and make a product. From this bacterial clone, the cloned gene can

be isolated for further analysis, or protein product can be separated (Fig 23.2). Besides

plasmids, the DNA of bacterial viruses (for example, lambda phage) can also be used

as a vector. After lambda phage attaches to a host bacterium, recombinant DNA is

released from the virus and enters the bacterium. Here, it will direct the reproduction

of many more viruses. Each virus in bacteriophage clone contains a copy of the gene

being cloned (Fig. 23.3).

Fig. 23.3 Plasmid DNA (upper part of igure) as well as viral DNA (lower part of the igure) can be used as vectors for cloning gene of interes

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Genoiftic Library

A genome is a full set of genes of an individual. A genomic library is a collection of

bacterial or bacteriophage clones, each clone containing a particular segment of

DNA from the source cell. For making a genomic library, an organism’s DNA is simply

sliced up into pieces, and pieces are put into vectors (i.e. plasmids or viruses) that are

taken up by host bacteria as shown in Fig. 23.3. The entire collection of bacterial or

bacteriophage clones that result contains all the genes of that organism.

A particular probe can be used to search a genetic library for a certain gene. A probe

is a single stranded nucleotide sequence that will hybridize (pair) with a certain piece

of DNA. Location of the probe is possible because the probe is either radioactive or

luorescent. Bacterial cells, each carrying a particular DNA fragment, can be plated onto agar in a petri dish. After the probe hybridizes into the gene of interest, the genes can

be isolated from the fragment (Fig 23.4). Now this particular fragment can be cloned

further or even analyzed for its particular DNA sequence.

Fig 23.4 Identiication of a cloned gene

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Fig 23.5 Polymerase chain reaction (PCR)

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The polymerase Chain Reaction

Kary B. Mullis developed the polymerase chain reaction (PCR) in 1983. Earlier methods

of obtaining multiple copies of a speciic sequence of DNA were time consuming and expensive. In contrast, PCR can create millions of copies of a single gene or any speciic piece of DNA quickly in a test tube. PCR is very speciic - the targeted DNA sequence can be less than one part in a million of the total DNA sample. .This means that a single

gene or smaller piece of DNA, among all the human genes can be ampliied (copied) using PCR.

PCR takes its name from DNA polymerase, the enzyme that carries out DNA replication

in a cell. It is considered a chain reaction because DNA polymerase will carry out

replication over and over again, until there are millions of copies of the desired DNA.

PCR does not replace gene cloning, which is still used whenever a large quantity of

gene or protein product is needed.

Before carrying out PCR, primers - sequences of about 20 bases that are complementary

to the bases on either side of the “target DNA” - must be available. The primers are needed

because DNA polymerase does not start the replication process; it only continues or

extends the process. After the primers bind by complementary base pairing to the

DNA strand, DNA polymerase copies the target DNA (Fig 23.5) .

DNA polymerase used is temperature - insensitive (thermostable) enzyme extracted

from the bacterium Thermus aquaticus, which lives in hot springs. Commonly, this

enzyme is also known as Taq polymerase. It can withstand high temperature, which is

used to separate double stranded DNA, therefore, replication need not be interrupted

by the need to add more enzyme. PCR is done these days in an automatic PCR machine

or thermocycler, which is a routine piece of equipment in any laboratory.

Animation 23.2 : PCR

Source & Credit: members.jcom

http://members.jcom.home.ne.jp/kisono/pcr/pcr.htm

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Analyzing DNA

The entire genome of an individual can be subjected to DNA inger printing, a process described in Fig. 23.6. The genome is treated with restriction enzymes, which results

in a unique collection of diferent sized fragments. Therefore, restriction fragment length polymorphism (RFLPs) exists between individuals. During a process called gel

electrophoresis, the fragments can be separated according to their lengths (molecular

weight or size), and the result is a number of bands that are so close together that

they appear as a smear. However, the use of probes for genetic markers produces a

distinctive pattern that can be recorded on X-ray ilm.

Fif 23.6 DNA ingerprinting. Three samples of DNA(I , II , IIi)were cut with a restriction enzyme and run on agarose gel. The gel pattren was then transferred to a membrane and DNA was denatured. The denatured DNA on the paper was hybridized with radioactive probe. Since the radioactive probes and complemetary arrangement of bases to the original DNA ,all DNA

fragments were labelled , which appeared as black bands with autogradiagram.

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The DNA from a single sperm enough to identify a suspected rapist. Since DNA

is inherited, its inger print resembles that of one’s parents. DNA inger printing successfully identiied the remains of a teenager who had been murdered eight years before because the skeletal DNA was similar to that of the parent’s DNA.

In Fig. 23.7 are given some DNA inger prints. The igure 23.7 (a) shows comparison of child’s inger print with that of his parents. The child has received DNA from both of his parents. Arrows indicate that some bands in him are like his father, some like his

mother. Some bands are. however, unique to him, which do not match with any of the

parents.

Fig. 23.7 (b) shows a case of disputed parenthood. Two persons F1 and F

2 claim to be the

father of child C, whose mother’s inger print is given under M. The child has received DNA from both of his parents. Obviously F

1is not the real father.

The arrows on left side show common bands between mother and child while those on

right show common bands between the father and the child.

Fig. 23.7(c) shows DNA inger prints which have been presented as forensic evidence. A criminal on a deserted place assaulted a woman. She scratched his face in her defence

but he murdered her and ran away. Forensic scientist recovered murder’s hair and skin

cells from underneath her nails. They prepared DNA inger prints from blood of victim, from murderer’s skin and hair, and from three suspects blood. Can you compare them

for speciic DNA sequence and tell who is in guilty and who is not? The suspect 1 has inger prints, which is similar to linger print from skin cells taken from underneath nails of the victim.Therefore suspect 1 is the culprit. The suspect 2 and 3 are not.

PCR ampliication and analysis can be used (1) to diagnose viral infections, genetic disorders, and cancer (2) in forensic laboratories to identify criminals; and (3) to

determine the evolutionary history’ of human population. It has been possible to

sequence DNA taken from a 76,000 years old mummiied human braiti and from a 17 to 20 million years old plant fossil following PCR ampliication.

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Fig 23.7(a) Comparison of a child’s DNA ingerprint (c) with his parent’s DNA

fringerpints (Mand F),

(b) DNA ingerprints as evidence for paternity. (c) DNA Test - a powerful tool of forensic science.

Gene Sequencing

In the late 1970s, methods were developed that allowed the nucleotide sequence of

any puriied DNA fragement to be determined simply and quickly. The main principle of these methods is :

1. To generate pieces of DNA of diferent sizes all starting from the same point and ending at diferent points.

2. Separation of these diferent pieces of DNA on agarose gel. 3. Reading of sequence from the gel.

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For generation of diferent sized DNA fragments, two methods arc generally used. One is Sanger’s,method in which dideoxyribonucleoside triphosphates arc used to terminate

DNA synthesis at diferent sites. The other method is known as Maxam-Giibcrt method in which DNA threads are chemically cut into pieces of diferent sizes

Fig 23.8 The enzymatic or dideoxy method of sequencing DNA

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Fig 23.8 shows typical gel obtained after dideoxy method. The volume of DNA sequence

information is now so large that powerful computers must be used to store and analyze

it. DNA sequence is now completely automated, robotic devices mix the reagents and

then load, run and read the order of the nucleotide bases from the gel. This is facilitated

by using chain terminating nucleotides that are each labelled with a diferent colored luorescent dye; in this case, all four synthesis reactions can be performed in the same tube, and the products can be separated in a single lane of a gel. A detector positioned

near the bottom of the gel reads and records the color of luorescent label on each band as it passes through a laser beam. A computer then reads and stores this nucleotide

sequence.

Owing to the automation of DNA sequencing, the genomes of many organisms have

been sequenced. These include plant chloroplasts and animal mitochondria, large

number of bacteria, many of the yeasts, a nematode worm. Drosophila, the model plant

Arabidopsis, the mouse and human. Researchers have also deduced the complete DNA

sequence of a variety of human pathogens.

THE HUMAN GENOME PROJECT

The human genome project is massive efort originally founded by the U.S. govern-

ment and now increasingly by U.S. pharmaceutical companies to map the human

chromosomes. Many non-proit and for proit biochemical laboratories around the world are now involved in the project which has two primary goals.

Animation 23.3: Human Genome Project

Source & Credit: CRDD

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Fig 23.9 Genetic map of X chromosome

The irst goal is to construct a genetic map of the human genome. The aim is to show the sequence of genes along the length of each type of chromosome, such as de-

picted for the X chromosome in Fig 23.9. When the DNA sequence of human chro-

mosome no. 22, one of the smallest human chromosomes, was completed in 19.99,

it became possible for the irst time to see exactly how genes are arranged along an entire vertebrate chromosome. With the publication of the entire human genome in

2001, the genetic landscape of all human chromosomes suddenly came into sharp

focus. The sheer quantity of information provided by the human genome project is

unprecedented in biology. The human genome is 25 times larger than any other ge-

nome sequenced so far.

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The map for each chromosome is presently incomplete, and in many instances scientists

rely on the placement of RFLPs. These sites eventually allow scientist to pinpoint disease

causing genes because a particular RFLP and a defective gene are often inherited

together. For example it is known that persons with Huntington disease have a unique

site where a restriction enzyme cuts DNA. The test for Huntington disease relies on this

diference from the normal. The second goal is to construct a base sequence map. There are three billion base

pairs in the human genome and it is estimated it cohld take an encyclopaedia of 200

volumes, each with 1000 pages, to list all of these. Yet this goal has been reached and

all the chromosomes have been sequenced.

BIOTECHNOLOGY PRODUCTS

Today bacteria, plants and animals are genetically engineered to produce biotechnology

products. Organisms that have a foreign gene inserted into them are called transgenic

organisms.

Transgenic Bacteria Recombinant DNA technology is used to produce bacteria that reproduce in large

vats called bioreactors. If the foreign gene is replicated and actively expressed, a

large amount of protein product can be obtained. Biotechnology products produced

by bacteria, such as insulin, human growth hormone, tissue plasminogen activator,

haemophilia factor Vm, and hepatitis B vaccine are now in the market.

Transgenic bacteria have been produced to promote health of plants for example,

bacteria that normally live on plants and encourage the formation of ice crystals have

been changed from frost - plus to frost - minus bacteria. Also, a bacterium that normally

colonizes the roots of com plants has now been endowed with genes (from another

bacterium) that code for an insect toxin. The toxin protects the roots from insects.

Bacteria can be selected for their ability to degrade a particular substance and then

this ability can be enhanced by genetic engineering. For instance, naturally occurring

bacteria may be engineered to do an even better job of cleaning up beaches after oil

spills.

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Industry has found that bacteria can be used as bioilters to prevent airborne chemical pollutants from being vented into the air. They can also remove sulfur from coal before

it is burned and help to clean up toxic waste dumps. One such strain was given genes

that allowed it to clean up levels of toxins that would have killed other strains. Further,

these bacteria were given “suicide” genes that caused them to self-destruct when the

job had been accomplished.

Organic chemicals are often synthesized by having catalysts act on precursor molecules

or by using bacteria to carry out the synthesis. Today, it is possible to go one step further

and to manipulate the genes that code for these enzymes. For instance, biochemists

discovered a strain of bacteria that is specially good at producing phenylalanine; an

organic chemical needed to make aspartame, the dipeptide sweetener better known

as Nutrasweet. They isolated, altered and formed a vector for the appropriate genes

so that various bacteria could be genetically engineered to produce pucnylaianine.

Many major mining companies already use bacteria to obtain various metals. Genetic

engineering may enhance the ability of bacteria to extract copper, uranium and gold

from low grade sources. Some mining companies are testing genetically engineered

organisms that have improved bioleaching capabilities.

Animation 23.4: Transgenic Bectria

Source & Credit: 33rd Square

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Transgenic Plants

Techniques have been developed to introduce foreign genes into immature plant

embryos, or into plant cells that have had the cell wall removed and are called

protoplasts. It is possible to treat protoplasts with an electric current while they are

suspended in a liquid containing foreign DNA. The electric current makes tiny, selfscaling

holes in the plasma membrane through which genetic material can enter. Then a

protoplast will develop into a complete plant. Foreign genes transferred to cotton, com

and potato strains have made these plants resistant to pests because their cells now

produce an insect’toxin. Similarly, soybeans have been made resistant to a common

herbicide. Some corn and cotton plants are both pest and herbicide resistant. In 1999

these transgenic crops were planted on more than 70 million acres worldwide and the

acreage is expected to triple in about ive years. Improvements still to come for are increased protein or starch content and modiied oil or amino acid composition.

Animation 23.5:Transgenic Plants

Source & Credit: Wikipedia

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Agribusiness companies also are in the process of developing transgenic versions

of wheat and rice in addition to com. This is considered an absolute necessity if the

2020 global demand for rice, wheat and com is to be met. World grain harvests have

continued to rise since the 1960s when special high-yield hybrid plants were developed

during the so called green revolution. But the per capita production has now lattened out because of continued population growth. The hope is that genetic engineering will

allow fanners to surpass the yield barrier. Perhaps, the stomata, the pore-like openings

in the leaves, could be altered to boost carbon dioxide intake or cut down water loss.

Another possible goal is to increase the eiciency of the enzyme Rubisco which captures C02 in most plants. A team of Japanese scientists are attempting to introduce the C4

cycle into the rice. Plants that utilize the C4 cycle avoid the ineiciency of carboxylase by using a diferent means of capturing C02. Unlike the single gene transfers that have been done so far, these modiications would require a thorough re-engineering of plant cells. Single gene transfers will cause plants to produce various products. A weed

called mouse-eared cress has been engineered to produce a biodegradable plastic

(polyhydroxy-butyrate) in cell granules.

Plants are being engineered to produce human hormones, clotting factors, and

antibodies in their seeds. One type of antibody made by com can deliver radio

isotopes to tumor cells, and another made by soybeans can be used as treatment for

genital herpes. Plant-made antibodies are inexpensive and there is little worry about

contamination with pathogens that could infect people. Clinical trials have begun.

Transgenic Animals

Techniques have been developed to insert genes into the eggs of animals. It is possible

to micro eggs by hand, but another method uses vortex mixing. The eggs and silicon -

carbide needles, and the needles make DNA can enter. When these eggs are fertilized,

the resulting ofspring are transgenic animals. Using this technique many types of animal eggs have taken up the gene for bovine growth hormone. The procedure has been

used to produce larger ishes’, cows, pigs, rabbits and sheep. Genetically engineered ishes are now being kept in ponds that ofer no escape to the wild because there is much concern that they will upset or destroy natural ecosystems.

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(a) This goat is genetically engineered to produce antithrobin III , which is secreted in her milk

(b) The procedure to produce a transgenic animal.

(c) The procedure to clone a transgenic animal

Gene pharming, the’ use of transgenic farm animals to produce pharmaceuticals is

being pursued by a number of irms. Genes that code for therapeutic, and diagnostic proteins are incorporated into the animal’s DNA, and the proteins appear in the animal’s

milk. There are plans to produce drugs for the treatment of cystic ibrosis, cancer, blood diseases and other disorders. Antithrombin III, for preventing blood clot during surgery,

is currently being produced by a herd of goats, and clinical trials have begun. Figure

23.10 out lines the procedure of producing transgenic mammals. DNA containing the

gene of interest is injected into donor eggs. Following in vitro fertilization, the zygotes

are placed in host females where they develop. After female ofspring mature, the product is secreted in the milk. The scientists of United States Department

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of Agriculture have been able to genetically engineer mice to produce human growth

hormone in their urine instead of in milk. They expect to be able to use the same

technique on larger animals. Urine is a preferable vehicle for a biotechnology product

than milk because all animals in a herd urinate - only females produce milk; animals

start to urinate at birth - females don’t produce milk until maturity; and its easier to

extract proteins from urine than from milk.

Cloning of Transgenic Animals

Imagine that an animal has been genetically engineered to produce a biotechnology

product. What would be the best possible method of getting identical copies of the

animals? Asexual reproduction through cloning the animal would be the preferred procedure to use. Cloning is a form of asexual reproduction because it requires only

the genes of that one animal. For many years it was believed that adult vertebrate

animals could not be cloned. Although each cell contains a copy of all the genes certain

genes are turned of in mature specialized cells. Diferent genes are expressed in muscle cells, which contract, compared to nerve cells, which conduct nerve impulses

and to glandular cells, which secrete. Cloning of an adult vertebrate requires that all

genes of an adult cells be turned on again if development is to proceed normally. It had

long been thought this would be impossible. In 1997, scientists at the Roslin Institute

in Scotland announced that they achieved this feat and had produced a cloned sheep

called Dolly.

Since then calves and goats have been cloned. Figure 23.10 shows that after

enucleated eggs have been injected with 2n nuclei of adult cells, they can be

coaxed to begin development. The ofspring have the genotype and phenotype of the adult that donated the nuclei; therefore, the adult has been cloned. In the

procedure that produced cloned mice, the 2n nuclei were taken from cumulus cells.

Cumulus cells are those that cling to an egg after ovulation occurs. A specially prepared

chemical bath was used to stimulate the eggs to divide and begin development. Now

that scientists have a method to clone mammals, this procedure will undoubtedly

be used routinely. In the United States, a presidential order prohibits the cloning of

humans. But certain other countries are experimenting with the possibility.

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GENE THERAPY

Gene therapy is the insertion of genetic material into human cells for the treatment

of a disorder. It includes procedures that give a patient healthy genes to make up for

faulty genes and also includes the use of genes to treat various other human illnesses

such as cancer and cardiovascular diseases.

There are two main methods used for gene therapy Ex-vivo and in vivo. Ex- vivo

gene therapy is shown in Fig. 23.11. in which children in the severe combined

immunodeiciency syndrome (SCID) is treated. These children lack an enzyme adenosine deaminase (ADA) that is involved in the maturation of T and B cells and,

therefore, they are subjected to life threatening infections. Bone marrow stem cells

are removed from the blood and infected with a retrovirus (RNA virus) that carries a

normal gene for the enzyme then the cells are returned to the patient. Bone marrow

stem cells are preferred for this procedure, because they divide to produce more cells

with same genes. Patients who have undergone this procedure do have a signiicant improvement in their immune function that is associated with a sustained rise in the

level of ADA enzyme activity in the blood

Animation 23.6: Gene TherapySource & Credit: Ethris

gfrjtj

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Among the many gene therapy trials, one is for the treatment of familial

hypercholesterolemia a condition that develops when liver cells lack a receptor for

removing cholesterol from the blood. The high levels of blood cholesterol make the

patient subject to fatal heart attacks at a young age. In a newly developed procedure, a

small portion of the liver is surgically excised and infected with a retrovirus containing

a normal gene for the receptor. Several patients have experienced a lowering of serum

cholesterol levels following this procedure.

Fig 23.11 Ex vivo gene therapy in human

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Cystic ibrosis patients lack a gene that codes for trans-membrane carrier of the chloride ion. Patients often die due to numerous infections of the respiratory tract.

And in vivo method of treatment is being tried. Liposomes-microscopic vesicles that

spontaneously form when lipoproteins are put into a solution, have been coated with

the gene needed to cure cystic ibrosis. Then the solution is sprayed into patient’s nostrils. Due to limited gene transfer, this methodology has not as yet been successful.

Gene therapy is also being done to cancer patients, which makes them more tolerant

of chemotherapy. In clinical trials researchers have given genes to cancer patient

that either make healthy cells more tolerant of chemotherapy or make tumors more

vulnerable to it. Once the bone marrow stem cells were protected it was possible to

increase the level of chemotherapy to kill the cancer cells.

During coronary artery angioplasty, a balloon catheter is sometimes used to open

up a closed artery. Unfortunately, the artery has a tendency to close up once again.

But investigators have come up with a new procedure. The balloon is coated with a

plasmid that contains a gene for vascular endothelial growth factor. The expression of

the gene, which promotes the proliferation of blood vessels to bypass the obstructed

area, has been observed in at least one patient.

Perhaps it will be possible to used in vivo therapy to cure hemophilia, diabetes.

Parkinson disease, or AIDS. To treat hemophilia, patients could get regular doses of cells

that contain normal clotting-factor genes or such cells could be placed in organoids,

artiicial organs that can be implanted in the abdominal cavity. To cure Parkinson’s disease, dopamine-producing cells could be grafted directly into the brain.

TISSUE CULTURE

Tissue culture is the growth of a tissue in an artiicial liquid culture medium. German botanist Gottlieb Haberlandt said in 1902 that plant cells are totipotent - each cell has

the full genetic potential of the organism - and, therefore, a single cell could become

a complete plant. But it wasn’t until 1958 that Cornell botanist F.C. Steward grew a

complete carrot plant from a tiny piece of phloem. He provided the cells with sugars,

minerals and vitamins, but he also added coconut milk. (Later it was discovered that

coconut milk contains the plant hormone cytokinin). When the cultured cells began

dividing, they produced a callus, an undiferentiated group of cells.

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Then the callus diferentiated into shoot and roots and developed into a complete plant.

Tissue culture techniques have by now led to micropropagation, a commercial method

of producing thousands, even millions of identical seedlings in a limited amount of

space. One favourite method to accomplish micro propagation is by meristem culture.

If the correct proportions of auxins and cytokinin are added to a liquid medium, many

new shoots will develop from a single shoot tip. When these are removed more shoots

form. Since the shoots are genetically identical the adult plants that develop from them

are called clonal plants, all having the same traits. Another advantage of meristem

culture is that meristem, unlike other portions of a plant, is virus free, therefore the

plants produced are also virus free (The presence of plant viruses weakens plants and

makes them less productive).

Because plants are totipotent, it should be possible to grow an entire plant from

a single cell. This, too has been done. Enzymes are used to digest the cell walls of a

small piece of tissue, usually mesophyll tissue, from a leaf, and the result is naked

cells without walls, called protoplasts. The protoplasts regenerate a new cell wall

and begin to divide. These clumps of cells can be manipulated to produce somatic

embiyos. Somatic embryos that are encapsulated in a protective hydrated gel (and

sometimes called artiicial seeds) can be shipped everywhere. It is possible to produce millions of somatic embryos at once in large tanks called bioreactors. This is done for

certain vegetables like tomato, celery, asparagus and for ornamental plants like lilies,

begonias and African violets. A mature plant develops from each somatic embryo.

Plants generated from the somatic embryo vary somewhat because of mutations that

arise; dunng the production process. These so called somaclonal variations are another

way to produce new plants with desired traits.

Anther culture is a technique in which mature anthers are cultured in a medium

containing vitamins and growth regulators. The haploid tube cells with in the pollen

grains divide, producing proembryos consisting of as many as 20 to 40 cells. Finally the

pollen grains rupture releasing haploid embryos. The experimenter can now generate a

haploid plant, or chemical agent can be added that encourages chromosomal doubling.

After chromosomal doubling the resulting plants are diploid but homozygous for all

their alleles. Anther culture is a direct way to produce plants that express recessive

alleles. If the recessive alleles govern desirable traits, the plants have these traits.

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The culturing of plant tissues has led to a technique called cell suspension culture.

Rapidly growing cultures are cut into small pieces and shaken in’a liquid nutrient medium

so that single cells or small clumps of cells break of and form a suspension. These cells will produce the same chemicals as the entire plant. For example cell suspension

cultures of Cinchona ledgeriana produce quinine and those of Digitalis lanata produce

digitoxin. Scientists envision that it will be possible to maintain cell suspension cultures

in bioreactors for the purpose of producing chemicals used in the production of drugs,

cosmetics and agricultural chemicals. If so, it will no longer be necessary to farm plants

for the purpose of acquiring the chemicals they produce.

Genetic Engineering of Plants

Traditionally, hybridization, the crossing of diferent varieties of plants or even species, was used to produce plants with desirable traits. Hybridization, followed by vegetative

propagation of the mature plants, generated a large number of identical plants with

these traits. Today it is possible to directly alter the genes of organisms. Transgenic

plants carry a foreign gene that has been introduced into their cells so that they have

new and diferent traits.

Since a whole plant will grow from a protoplast, it is necessary only to place the foreign

gene into a living- protoplast. A foreign gene isolated from any type of organism is

placed in the tissue culture medium.

High-voltage electric pulses can then be used to create pores in the plasma membrane

so that the DNA enters. In one of the irst procedures carried out, a gene for the production of the irely enzyme luciferase was inserted into tobacco protoplast and the adult plants glowed when sprayed with the substrate luciferin (Fig. 23.12).

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Unfortunately, the regeneration of cereal grains from protoplasts has been diicult. Com and wheat protoplasts produce infertile plants. As a result, other methods are

used to introduce DNA into plant cells with intact cell wall. In one technique, foreign

DNA is inserted into the plasmid of the bacterium Agrobacterium, which normally infects

the plant cells. A plasmid can be used to produce’ recombinant DNA. Recombinant

DNA contains genes from diferent sources, namely those of plasmids and the foreign genes of interest. When the bacterium infects the plant the recombinant plasmid is

introduced into the plant cells (Fig.23.12). In 1987, John C Sanford and Theodore M.

Klein of Cornell University developed another method of introducing DNA into a plant

tissue culture callus.

Fig 23.12 Tabacoo plant containing Luciferase gene glows when sprayed with luciferin

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They constructed a device, called the particle gun, that bombards a callus with DNA

coated microscopic metal particles. Then genetically altered somatic embryos develop

into genetically adult plants. Many plants including com and wheat varieties have been

genetically engineered by this method.

Agricultural Plants with Improved Traits

Cotton, com, potato and soybean plants have been engineered to be resistant to either

insect predation or herbicides that are judged to be environmentally safe. Some com

and cotton plants have been produced that are both insect and herbicide resistant.

In 1999, transgenic crops were planted on more that 70 million acres world wide and

the acreage is expected to triple in about ive years. If crops are resistant to a broad-spectrum herbicide and weeds are not then the herbicide can be used to kill the weeds.

When herbicide resistant plants were planted weeds were easily controlled, less tillage

was needed and soil erosion was minimized.

One aim of genetic engineering is to produce crops that have the improved agricultural

or food quality traits such as those listed in the table below:

Improved Agricultural Traits

Herbicide resistant Wheat, rice, sugar beets, canola

Salt tolerant Cereals, rice, sugarcane

Drought tolerant Cereals, rice, sugarcane

Cold tolerant Cereals, rice, sugarcane

Improved yield Cereals, rice, com, cotton

Modiied wood pulp Trees

Improved Food Quality Traits

Fatty acid / oil content Com, soybeans

Protein / starch content Cereals, potatoes, soybeans, rice, com

Amino acid content Com, soybean

Disease protected Wheat, com, potatoes

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Production of salt tolerant plants had been a dream of genetic engineer. Recently

salt - tolerant Arabidopsis has been produced. For this the scientists irst identiied a gene coding for a channel protein that transports Na+ along with H+ across a

vacuole membrane. Isolating Na+ in a vacuole prevents it from interfering with plant

metabolism. Then, the scientists cloned the gene and used it to genetically engineer

plants that overproduce the channel protein. The modiied plants thrived when watered with a salty solution. Irrigation, even into fresh water, inevitably leads to a

salinization of soil that reduces crop yields. Today, crop production is limited by efects of salinization at about 50% of irrigated levels. The next step to solve this problem is

to produce salt - tolerant crops. It is believed that the production not only of salt - but

also drought and cold tolerant crops will reduce the need for added farm acreage by

increasing agricultural yields that will provide enough food for a world population

that is expected to nearly double by 2050.

Some progress has also been made to increase the food quality of crops. Soybeans

have been developed that mainly produce the monounsaturated fatty acid, oleic acid,

a change that may improve human health. These altered plants also produce vernolic

acid and ricinoleic acid, derivatives of oleic acid that can be used as hardenes in paints

and plastics. The necessary genes were derived from Vemonia and castor bean seeds

and were transferred into the soybean genomes.

Genetic Engineering is also expected to increase productivity. To that end, stomata

might be altered to boost carbon dioxide intake or cut down water loss. The eiciency of the enzyme RuBP carboxylase which captures C02 in plants could be improved. A

team of Japanese scientists is working on introduc ing the C4 photosynthetic cycle into

rice. Unlike C3 plants, C4 plants do well in hot dry weather. These modiications would require a more complete engineering of plant cells than the single gene transfers’ that

have been done so far.

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Production of Products

Single gene transfers have allowed plants to produce various products such as human

hormones, clotting factors and antibodies. One type of antibody made by com can

deliver radioisotopes to tumor cells and another made by soybeans can be used as

treatment for genital herpes clinical triats have begun.

Recently, a group of scientists from Biosource Technologies located in Vacaville,

California reported that they have been able to use the tobacco mosaic virus as a

vector to introduce a human gene into adult tobacco plants in the ield. Note that this technology by passes the need for tissue culture completely. Tens of grams of

a-galactosidase, an enzyme that can be used to treat a human lysosome storage

disease, were harvested per acre of tobacco plants. And it only took thirty days to

get tobacco plants to produce antigens to treat non-Hodgkin’s lymphoma after being

sprayed with a genetically engineered vims.

EXERCISE

Q.1. Fill in the blanks.

1. The use of polymerase chain reaction (PCR) creates a _________ of copies in a laboratory

test tube.

2. ___________free living organisms in the environment that have had a foreign gene

inserted into them.

3. __________known sequences of DNA that are used to ind complementary DNA strands; can be used diagnostically to determine the presence of particular gene.

4. ___________production of many identical copies of a gene.

5. ___________self duplicating ring of accessory DNA in the cytoplasm of bacteria.

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Q.3. Short questions.

1. How and why transgenic animals that secrete a product are often cloned? 2. Explain two primary goals of Human Genome Project. What are possible beneits of

the project? 3. Explain and give examples of ex vivo and in vivo gene therapies in humans?

Q.4. Extensive questions.

1. What is the methodology for producing recombinant DNA to be used in gene cloning?2. What is a genomic library, how would you locate a gene^of.interest in the library? 3. What is the polymerase chain reaction (PCR), amftow is it carried out to produce

multiple copies of a DNA segment? 4. What is DNA inger printing, a process that utilizes the entire genome? 5. For what purpose have bacteria, plants and animals been genetically altered?-

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